Abstract: Adiantum nelumboides X. C. Zhang, belonging to the family Pteridiaceae, is an evergreen plant with a solitary frond, endemic to a restricted range within the Wanzhou and Shizhu counties of Chongqing, China. Owing to its narrow distribution and declining populations, it is classified as a rare and threatened species. Optimizing in vitro propagation and enhancing spore yield represent key strategies for its conservation. However, systematic studies on the regulation of its sexual reproductive process remain limited. Here, an in vitro culture system was employed to investigate the effects of calcium ions (Ca²⁺), plant growth regulators, and medium status on sexual reproduction in A. nelumboides. Results showed that supplementation with Ca²⁺ promoted the development of gametophytic meristems and archegonia, and induced the formation of 3–5 sporophytes from a single gametophyte. However, prolonged Ca²⁺ exposure suppressed further sporophyte differentiation. Combined application of BA and NAA enhanced antheridia differentiation and increased archegonial density, and induced the production of tetraploid sporophytes. However, extended exposure to these growth regulators inhibited sporophyte formation. Gametophytes maintained in liquid media or under isolated culture conditions exhibited a shift toward vegetative proliferation, often losing the capacity to form multiple sporophytes. This reproductive potential was partially restored upon transfer to solid culture or when cultured together. Collectively, these findings demonstrate that sexual reproduction in A. nelumboides is modulated by Ca²⁺ availability, plant growth regulators, culture substrate, and gametophyte density, with Ca²⁺ and plant growth regulators exerting temporally opposing effects on sporophyte development.